What spatial structure and mechanics develops in biofilm infections, and how such spatial structure and mechanics impact the persistence and virulence of biofilm infections, is not known. The long-term goal is to find diagnostic and treatment approaches that address the structure and mechanics of multicellular, three- dimensional biofilm infections within the host. The objective of this proposal is to determine the mechanics and structure of biofilm infections of the opportunistic pathogen Pseudomonas aeruginosa in chronic wounds, and how these physical properties impact disease course. The central hypothesis is that spatial structure and mechanics are the major physical factors controlling virulence, antibiotic resistance, and immune evasion in biofilm infections. The rationale underlying this proposal is that completion will identify key physical targets for preventing, disrupting, or ameliorating biofilm infections for an important biofilm-forming pathogen. The proposed work will also develop a widely-applicable platform for assessing the state and impact of biofilm structure and mechanics for other infecting organisms. The central hypothesis will be tested by pursuing three specific aims: 1) Determine the spatial structure and mechanics of in vivo biofilm infections; 2) Determine how spatial arrangements differentiate into distinct microenvironments; 3) Determine the role of spatial structure and mechanics in biofilm-neutrophil interactions. We will pursue these aims using an innovative combination of analytical and manipulative techniques from both biological and physical sciences. These include both recently-developed techniques specific to biofilm studies, and more-established techniques that have been applied very little to the study of biofilm materials. The proposed research is significant, because it will determine which structural and mechanical characteristics should be therapeutic targets. It is also significant because it will develop a platform that can be extended to study other pathogens (or commensals) and synergies to open new avenues for biofilm therapies. This work will develop foundational resources that will be used by other researchers, for P. aeruginosa and other organisms. The proximate expected outcome of this work an understanding of which biofilm structural and mechanical characteristics contribute to clinical impact. The results will have an important positive impact immediately because they will establish better understanding of biofilm infection, virulence, and resistance to antibiotics and the immune system for an important pathogens, and long-term because they lay the groundwork to develop a suite of techniques for better treatment of biofilm infections.